The goal of the proposed study is to define new mechanisms that regulate phototransduction and adaptation in photoreceptor cells. The proposal focuses on one of the central reactions that determines the duration of the photoresponse, hydrolysis of GTP by transducin. Aim 1 is to continue quantitative analyses of the regulation of transducin GTPase by cGMP binding to the noncatalytic sites of the cGMP PDE. The extent of this regulation will be determined by measuring the rates of GTP hydrolysis by transducin in a complex with PDE with or without cGMP bound to the noncatalytic sites. The same regulatory mechanism will also be studied in cone photoreceptors. Practically nothing is yet known about the rate of transducin GTPase and the importance of its regulation by cGMP in cones. This is primarily due to the fact that a cone preparation has not been available for biochemical studies. This problem will now be approached with a recently developed cone preparation obtained from the retina of the striped bass. Aim 2 is to establish the physiological role of the cGMP-dependent regulation of transducin GTPase in photoreceptors. The light-dependent changes in cGMP binding to the PDE non-catalytic sites in intact photoreceptors will be analyzed in detail. These changes will then be correlated with the changes in kinetics and sensitivity of rod photoresponses in collaboration with physiologists. In aim 3, one of the most important unresolved problems in regulation of transducin GTPase will be addressed, i.e., identification of the membrane-associated factor which, in combination with the PDE inhibitory gamma subunit, acts as a GTPase-activating protein (GAP) for transducin. The experiments outlined in this application are relevant to understanding the intricate feedback controls that regulate photoreceptor activity, controls that may be perturbed in several inherited diseases.